The goal of this project is to generate robust, portable assay systems for the rapid detection of botulinum neurotoxin (BoNT) in clinical samples. Our objective is to detect active BoNT within a 45 min test time at a limit of detection that is at least 10-fold better than the current gold standard, the live mouse bioassay. BoNT is the most potent biological toxin known. When injected, inhaled or ingested in minute amounts, BoNT elicits flaccid paralysis in humans and other mammals. However, BoNT is also widely available and used as a medical drug for cosmetic and therapeutic purposes. The availability and potential abuse of BoNT products presents a real threat to public health, and is of great concern to our national security. Our approach is based on a bead-based prototype assay with a large immuno-sorbent surface area (ALISSA) that can detect attomolar concentrations of BoNT serotype A in complex biological matrices such as blood serum and liquid foods. The BoNT ALISSA is at least four orders of magnitude more sensitive than the live mouse bioassay and considerably faster, requiring only hours instead of days. It is also much more economical and doesn't require the use of laboratory animals. To establish proof-of concept and improve the BoNT ALISSA, we propose the following Specific Aims: 1. To generate affinity reagents for microcolumn-capture of BoNT serotypes A and B (including subtypes). 2. To engineer novel substrates for BoNT-triggered dual-step enzyme cascades with bioluminescent readout for use in the ALISSA. 3. To generate affinity reagents for BoNT ALISSA detection of serotypes C, E, and F. 4. To establish the BoNT ALISSA system for serotypes A, B, C, E and F at a state laboratory of the Biodefense Laboratory Response Network for preclinical data collection on samples of infant botulism. The long term goal of the project is to obtain regulatory approval for use of the microcolumn BoNT ALISSA as a diagnostic medical device in form of an Investigational Device Exemption. The anti-BoNT affinity reagents proposed here will be generated as recombinant antibodies in form of single chain fragment variables or antigen-binding fragments and mounted onto microaffinity columns that we place into the tips of disposable plastic pipettes. The columns will be capable of immuno-capturing BoNTs in complex matrices with high affinity and specificity. Bioluminescent substrates will be produced as genetically- engineered inactive versions of luciferases that are activated by BoNTs in the microcolumn-based ALISSA. The product of this research will be an easy to use kit of biochemical reagents and affinity pipette tips that will allow rapid, sensitive BoNT detection without the need for special laboratory equipment. Used as diagnostic tools, such kits should improve public health and enhance our national biodefense preparedness.